Structure and Dynamics of a Stabilized Coiled-Coil Domain in the P-TEFb Regulator Hexim1

The positive transcription elongation factor P-TEFb mediates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb is negatively regulated by the cellular protein Hexim1 (hexamethylene bisacetamide-inducible protein 1), which is highly conserved in higher eukaryotes. The C-terminal coiled-coil domain of Hexim1 recognizes the Cyclin T subunit of P-TEFb, whereas a central PYNT motif is required to inhibit the cyclin-dependent kinase Cdk9 by a yet unknown mechanism. Here, the crystal structure of the Cyclin T-binding domain (TBD) of human Hexim1 was determined at 2.1 Å resolution using a deletion mutant of three residues in its central stammer motif. The structure showed a continuous parallel coiled-coil domain of nine hepta-repeats with a preceding helix encompassing up to 15 residues. Two uncommon residues at heptad a positions in the N-terminal part of the coiled-coil structure, Lys284 and Tyr291, stabilize the preceding helix by a tight intermolecular hydrogen bond network with residues of the opposing chain. These interactions delineate a characteristic turn between both helices that is supposed to mediate binding to Cyclin T1. Stabilization of the coiled-coil domain by deletion of the stammer region was confirmed by NMR spectroscopic and backbone dynamic analyses analyzing wild-type TBD and three mutant variants. This study thus provides structural insights into the recognition of the regulator protein Hexim1 by P-TEFb and the modulation of coiled-coil dynamics by specific discontinuities. [Display omitted] ► Crystal structure of Hexim1-TBD at 2.1 Å resolution. ► Regulation of transcriptional elongation by inhibition of P-TEFb. ► Coiled-coil stabilization by deletion of a central stammer element. ► Identification of polar surfaces on Hexim1 and an evolutionarily conserved loop sequence. ► Implications of stutter and stammer elements on coiled-coil dynamics.